1. Field of the Invention
The invention relates to dopamine receptors from mammalian species and the genes corresponding to such receptors. In particular, it relates to the human dopamine receptor D4. Specifically, the invention relates to the construction of recombinant expression constructs capable of expressing the human D4 dopamine receptor in cultures of transformed eukaryotic cells and the production of human D4 dopamine receptor protein in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells producing the human D4 dopamine receptor for the characterization of antipsychotic drugs. 2. Description of the Related Art
Dopamine is a neurotransmitter that participates in a variety of different functions mediated by the nervous system, including vision, movement, and behavior (see generally Cooper et al., 1978, The Biochemical Basis of Neuropharmacology, 3d ed., Oxford University Press, New York, pp. 161-195). The diverse physiological actions of dopamine are in turn mediated by its interaction with one of the five known types of G protein-coupled receptors (D 1, D2, D3, D4 and DS), which either stimulate or inhibit the enzyme adenylyl cyclase in response to dopamine binding (Kebabian & Calne, 1979, Nature 277: 93-96). Alterations in the number or activity of these receptors may be a contributory factor in human disease such as Parkinson's disease (a movement disorder) and schizophrenia (a behavioral disorder).
A great deal of information has accumulated on the biochemistry of the D1 and D2 dopamine receptors, the archetypal dopamine receptors and the first to be studied, and methods have been developed to solubilize and purify these receptor proteins (see Senogles et al., 1986, Biochemistry 25: 749-753; Sengoles et al., 1988, J. Biol. Chem. 263: 18996-19002; Gingrich et al., 1988, Biochemistry 27: 3907-3912); said methods have been adapted to study the other types of dopamine receptors as well.
The D1 dopamine receptor in several tissues appears to be a glycosylated membrane protein of about 72 kD (Amlalky et al., 1987, Mol. Pharmacol. 31: 129-134; Niznik et al., 1988, Biochemistry 27: 7594-7599). The D2 receptor has been suggested to have a higher molecular weight of about 90-150 kD (Amlalky & Caron, 1985, J. Biol. Chem. 260: 1983-1986; Amlaiky & Caron, 1986, J. Neurochem. 47: 196-204; Jarvie et al., 1988, Mol. Pharmacol. 34: 91-97). A recently discovered additional dopamine receptor, termed D3 (Sokoloffet al., 1990, Nature 347: 146-151) has been shown to be expressed via an alternatively spliced mRNA, to produce proteins differing by the presence or absence of a 21 amino acid portion of the third cytoplasmic domain (Fishburn et al., 1993, J. Biol. Chem. 268: 5872-5878). Boundy et al. have used a baculovirus expression system to produce sufficient D3 protein to raise antibodies useful in immunoprecipitation and immunoblot assays of D3 receptor protein (1993, J. Pharmacol. Exper. Therap. 264: 1002-1011).
Dopamine receptors are primary targets in the clinical treatment of psychomotor disorders such as Parkinson's disease and affective disorders such as schizophrenia (Seeman et al., 1987, Neuropsychopharm. 1: 5-15; Seeman, 1987, Synapse 1: 152-333). The different dopamine receptors have been cloned as a result of nucleotide sequence homology between these receptor genes (Bunzow et al., 1988, Nature 336: 783-787; Grandy et al., 1989, Proc. Natl. Acad. Sci. USA 86: 9762-9766; Dal Toso et al., 1989, EMBO J. 8: 4025-4034; Zhou et al., 1990, Nature 346: 76-80; Sunahara et al., 1990, Nature 346: 80-83; Sokoloff et al., 1990, Nature 347: 146-151; Van Tol et al., 1991, Nature 350: 610-614; Van Tol et al., 1992, Nature 358: 149-152; Sunahara et al., 1991, Nature 350: 614-619).
The antipsychotic clozapine is useful for socially withdrawn and treatment-resistant schizophrenics (see Kane et al., 1990, Nature 347: 146-151), but unlike other antipsychotic drugs, clozapine does not cause tardive dyskinesia (see Casey, 1989, Psychopharmacology 99: 547-553). Clozapine cannot exert its effects via the D2 or D3 receptors, however, because the dissociation constants of D2 and D3 for clozapine are 3 to 30-fold higher than the therapeutic free concentration of clozapine in plasma water (Ackenheil et al., 1976, Arzneim-Forsch 26: 1156-1158; Sandoz Canada, Inc., 1990, Clozaril: Summary of preclinical and clinical dam). This observation suggested the existence of dopamine receptors more sensitive to the antipsychotic clozapine than D2 or D3.
Some of the present inventors have isolated and characterized the gene for the only known clozapine-reponsive human dopamine receptor, D4 (see U.S. patent application Ser. Nos. 07/928,611 and 07/626,618, both incorporated by reference). The human D4 dopamine receptor gene displays a high degree of homology to the human dopamine D2 and D3 receptor genes. The pharmacological profile of the receptor protein encoded by and produced by expression of this gene also resembles the D2 and D3 receptors, but has 10-fold higher affinity for clozapine.
Some of the same present inventors have also discovered that the D4 gene is polymorphic in the human population, having at least 7 different alleles that can be detected by restriction fragment length polymorphism analysis (see, Botstein et al., 1980, Am. J. Hum. Genet. 32: 314-331). This is the first human receptor gene in the catecholamine receptor family which displays such polymorphic variations in the coding region. The observed polymorphism in dopamine D4 receptor genes may underlie individual differences in susceptibility to neuropsychiatric disorders such as schizophrenia and manic depression, as well as responsiveness to antipsychotic medication.
Expression of these varying alleles of this clozapine-sensitive dopamine receptor protein receptor provides a useful method for screening putative psychotopic drugs in vitro to enable the discovery of new types of drugs for treatment of human diseases such as schizophrenia, which may share clozapine's useful and advantageous properties of not inducing tardive dyskinesia and other motor side effects.